Mouse tumor tissues were isolated, formalin fixed, paraffin embedded and cut into serial histologic sections, and stained with hematoxylin and eosin. Immunohistochemistry was performed with antibodies against cleaved-caspase3 (Wanlei, WL02117), FOXO1 (CST, #2880), Ki67 (Abcam, ab1667), α-SMA (Bioss, BS-10196R) and BCL-2 (Abclonal, A19693). TUNEL staining was performed with TUNEL label solution (Roche, G1501). DAPI was used for nuclear counterstaining for 10 min. Tissue sections from DLBCL patients were stained with BCL-2 (Abclonal, A19693), AGK (Invitrogen, PA5-28566) and FOXO1 (CST, #2880). Percentages of positive areas of different sections were determined by Image J.
Bcl 2
Manufactured by ABclonal
Sourced in China, United States, United Kingdom
Bcl-2 is a lab equipment product that functions as a regulator of apoptosis, or programmed cell death. It plays a critical role in the intrinsic apoptotic pathway, acting as an anti-apoptotic protein that can prevent or delay cell death. Bcl-2 is widely used in various research applications related to cell biology and biochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Bcl2 associated x (bax), by ABclonal (36 mentions)
Caspase 3, by ABclonal (16 mentions)
β actin, by ABclonal (13 mentions)
Gapdh, by ABclonal (12 mentions)
Caspase 9, by ABclonal (9 mentions)
Pvdf membrane, by Merck Group (7 mentions)
Cleaved caspase 3, by ABclonal (6 mentions)
Bca kit, by Thermo Fisher Scientific (6 mentions)
β actin, by Proteintech (5 mentions)
Caspase 8, by ABclonal (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Gapdh, by Proteintech (4 mentions)
N cadherin, by ABclonal (4 mentions)
Beclin1, by ABclonal (4 mentions)
E cadherin, by ABclonal (4 mentions)
Ripa buffer, by Beyotime (4 mentions)
Cyclin d1, by ABclonal (3 mentions)
Bca protein assay kit, by Beyotime (3 mentions)
Vimentin, by ABclonal (3 mentions)
Bcl xl, by ABclonal (3 mentions)
62 protocols using bcl 2
1
Immunohistochemical Analysis of Mouse Tumor Tissues
2
Western Blot Analysis of Cellular Proteins
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Total protein extraction from treated and untreated cells was performed using M-PER (including a protease inhibitor cocktail) followed by gel electrophoresis (SDS-PAGE) before being blotted onto a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The membrane was incubated in the blocking buffer (5% skimmed milk in TBST buffer), then treated with primary antibodies, including PDX1 (1:3000, #ab47267, Abcam, Cambridge, UK), BAD (A19595), BAX (A2211), PARP (A19596), AKT (#A17909), BCL-2 (#A19693), Phospho-AKT-S473 (#AP0637), and PI3Kinase p110 (#A19742), and from Abclonal technology (Woburn, MA, USA), Pro/Insulin (#81385), from Cell Signaling Technology (CA, USA) or β-actin (#A5441, Sigma-Aldrich, Hamburg, Germany) antibodies. Finally, the membrane was treated (for one hour at room temperature) with the secondary antibodies (#7076S and #7074S, Cell Signaling Technology, Santa Cruz, CA, USA). Chemiluminescence was detected using a Bio-Rad Enhanced chemiluminescence (ECL) substrate kit (Bio-Rad, Hercules, CA, USA). Protein bands were detected using Bio-Rad Image Lab software (Bio-Rad, Hercules, CA, USA). Image J software was used to calculate the band counts. As an endogenous control in all studies, β-actin was employed.
3
Isolation and Characterization of Wogonin
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Wogonin (purity ≥ 99%) is isolated from S. baicalensis Georgi according to previously reported protocols [31 (link)]. Wogonin was dissolved in dimethyl sulfoxide (DMSO) as a stock solution (100 mM) and stored at -80°C. The solution was freshly diluted with basal medium to designated concentrations, and the final concentration of DMSO will not be over 0.1%. Cells treated with the highest concentration of DMSO were used as the control in corresponding experiments. Navitoclax (CSN12932) was purchased from CSNpharm (Chicago, USA). Navitoclax powder was dissolved with DMSO to 10 mM and stored at -20°C. Primary antibodies against β-actin, GAPDH, trimethyl-histone H3-K9, BCL-2, hTERT, c-Myc, caspase-3, active caspase-3, p27, Bax, CHK2, cyclin D1, cyclin E1, CDK4, CDK6, HRP goat anti-mouse IgG (H+L), and HRP goat anti-rabbit IgG (H+L) were obtained from ABclonal Technology (Wuhan, China). Phospho-CHK2 (T68), phospho-p53 (Ser15), p21, and phospho-histone H2A.X (γ-H2A.X) were obtained from Cell Signaling Technology (Danvers, MA, USA). p53, p16, BCL-xL, Bim, and PARP-1 were obtained from Proteintech (CA, USA).
4
Protein Extraction and Western Blotting
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Total protein was extracted using RIPA lysate (Beyotime, Nantong, China) containing protease inhibitors and phosphatase inhibitors (CWBIO, Taizhou, China), and protein concentration was determined by the BCA protein assay kit (Beyotime, Nantong, China). Proteins were denatured in 5 × SDS-PAGE sample loading buffer (YEASEN, Shanghai, China), electrophoretically separated, and transferred onto PVDF membranes. After blocking in 5% skimmed milk for 2 h at room temperature, the proteins were incubated overnight at 4 °C with corresponding primary antibodies, including HSP90 (Proteintech, Wuhan, China), phospho-ERK1/2 (Cell Signaling Technology, Boston, MA, USA), ERK1/2 (Cell Signaling Technology, Boston, MA, USA), BCL2 (ABclonal, Wuhan, China), and BAX (ABclonal, Wuhan, China). Finally, the membranes were incubated in a horseradish peroxidase-labeled secondary antibody (CWBIO, Taizhou, China) for 2 h at room temperature. Protein expression was detected on a gel documentation system (Tanon, Shanghai, China) using ECL chemiluminescent chromogen solution (ABBKINE, Redlands, CA, USA).
5
Western Blot Analysis of Apoptosis and Oxidative Stress
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Proteins were extracted from brain tissues of the ischemic side and PC12 cells, separated on 10%–15% SDS-PAGE gels, and transferred to membranes. Then, the membranes were incubated with primary antibodies against Bax (1 : 1000, ABclonal, China), Bcl-2 (1 : 1000, ABclonal, China), caspase-3 (1 : 1000, ABclonal, China), cleaved caspase-3 (1 : 1000, ABclonal, China), Nrf2 (1 : 1000, Cell Signaling Technology, USA), HO-1 (1 : 1000, Proteintech, USA), Lamin B (1 : 1000, Abcam, USA), GAPDH (1 : 1000, Proteintech, USA), and β-actin (1 : 1000, Proteintech, USA). After three washes with TBST, the membranes were incubated with corresponding secondary antibodies (1 : 6500, Abcam, USA) and scanned using a BIO-RAD imaging system (BIO-RAD Gel Doc XR, USA). The band intensity was normalized to the intensity of the GAPDH or Lamin B band and analyzed using Image Lab v5.2 software.
6
Western Blot Analysis of Apoptosis Markers
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ESCC cells were plated into 6-well plates at a density of 2.5x105/well. After 24 hrs STA-9090 or vehicle was added into designated wells for 72h. The cells were lysed with RIPA Lysis Buffer and protein concentrations were measured with a BCA assay kit. The protein lysates were electrophoresed and transferred to PVDF membranes (GE Healthcare, Buckinghamshire, UK) and incubated overnight with the primary antibodies at 4°C. After washing with PBS the membranes were incubated with the appropriate special secondary antibodies at room temperature for 1h for signal detection. Antibodies used: HSP90 (1:1000) antibody and β-actin (1:10,000) antibody (Affinity Biosciences, OH, USA), BAX (1:500), BCL-2 (1:500), MYC (1:500) antibody (ABclonal Biotech Co, Woburn, MA, USA), Anti-mouse HRP (1:10,000) (ABclonal Biotech Co, Woburn, MA, USA), Anti-rabbit HRP (1:10,000) (ABclonal Biotech Co, Woburn, MA, USA).
7
Western Blot Analysis of Autophagy and EMT Markers
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Protein from cells or tissues was lysed using radioimmunoprecipitation assay buffer lysis buffer with 1% protease and phosphatase inhibitor cocktails (Beyotime Biotechnology). The western blotting (WB) assay was performed as previously described.33 Antibodies used in this experiment were as follows: UHMK1 (11624‐1‐AP; Proteintech), LC3 (A19665; ABclonal), Beclin1 (A21191; ABclonal), p62 (A19700; ABclonal), Bcl‐2 (A19 693; ABclonal), phosphoinositide 3‐kinase (PI3K) (A16950; ABclonal), protein kinase B (AKT) (A18675; ABclonal), phosphorylated AKT (p‐AKT) (AP1208; ABclonal), mammalian target of rapamycin (mTOR) (A11345; ABclonal), phosphorylated m‐TOR (p‐mTOR) (AP0115; ABclonal), E‐cadherin (A20798; ABclonal), N‐cadherin (A19083; ABclonal), vimentin (A19607; ABclonal), glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) (AC033; ABclonal). This experiment was repeated three times.
8
Western Blot Analysis of Signaling Pathways
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MTT, Trizma® base, glycine, SDS, acrylamide, N, N-Methylene bisacrylamide, and DMSO were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s Modified Eagle Medium (DMEM), phosphate buffer saline (PBS), and penicillin-streptomycin solution were from Hyclone (Logan, UT, USA). Fetal bovine serum (FBS) was purchased from Gibco (Grand Island, NY, USA). Doxorubicin hydrochloride was purchased from Aladdin (Shanghai, China). Rabbit polyclonal antibodies against STAT3, phospho-STAT3 (Tyr705), VEGF, MMP-7, Cyclin D1, Mcl-1, Survivin, Bcl-XL, Bcl-2, β-actin, and HRP-conjugated goat anti-rabbit antibody were purchased from ABclonal (Woburn, MA, USA). WesternBright™ Sirius chemiluminescent detection reagent was from Advansta (Menlo Park, CA, USA). Stock solutions of compounds (50 mM) were prepared in DMSO and stored at 4 °C. The stock solutions were diluted with DMEM to working concentrations as working solutions.
9
Western Blot Analysis of Apoptosis and Metastasis Markers
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HepG2 cells were lysed in RIPA buffer (containing 1 mmol·L−1 PMSF) (Beyotime, Shanghai, China). The protein samples were quantified using an enhanced BCA assay kit (Beyotime, Shanghai, China). Proteins were separated on a 10% or 15% SDS-PAGE gel by electrophoresis and then transferred to a 0.45 or 0.22 μm PVDF membrane (Millipore, Darmstadt, Germany). The membrane was blocked with 5% skim milk and incubated with primary antibodies for caspase-8, caspase-9, caspase-3, Bax, Bcl-2, Mcl-1, Bcl-XL, survivin, MMP-7, MMP-9, and β-Actin (ABclonal, Wuhan, China) overnight at 4 °C and washed with TBST buffer three times. We then incubated the samples with HRP-conjugated goat-anti-rabbit IgG secondary antibody (ABclonal, Wuhan, China) and then washed with TBST buffer three times. Finally, we applied 1 mL of ECL agent (Advansta, Menlo Park, CA, USA) on the membrane, and the protein blots were developed using an Omega Lum G imaging system (Aplegen, Pleasanton, CA, USA). Image J software was used to scan the gray values of the blots.
10
Western Blot Analysis of Protein Expression
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Experimental monolayers were washed with serum free media, and then total and fractionated proteins were extracted by cell lysis buffer (Cell Signaling Technology, Danvers, MA). The lysates were centrifuged at 12,000×g for 20 min at 4 °C. Equal amounts of protein, after concentration was determined by the Bradford assay (Bio-Rad, Hercules, CA), were loaded on SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad). After blocking, specific antibodies such as Bax, caspase-3, caspase-8, Bcl-2, PI3K, Notch1, p-NF-κB, NF-κB, E-cadherin, N-cadherin and β-actin from AB clonal Biotechnology Co., Ltd. (Wuhan, China) were used to perform detection. Finally each protein was detected using an enhanced chemilumi-nescence system (GE Healthcare, USA). Blot images were digitized (Chemidoc, Bio-Rad, Milan, Italy) and the area of each band was quantified using the computerized imaging system (QuantityOne, Bio-Rad). Relative optical density (arbitrary units) was normalized for control bands in each series and for protein loading (as probed by anti-actin blots). Each test was performed in triplicate experiments.
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